Noroviruses (NoVs) are the most common cause of epidemic gastroenteritis, responsible for at least 50% of all gastroenteritis outbreaks worldwide, and a major cause of food-borne illness. NoVs are a diverse group of viruses in the family Caliciviridae, and they are currently classified into six genogroups (GI–GVI) and further subdivided into multiple genotypes. Strains that infect humans are found in GI, GII, and GIV, with GII.4 strains notably causing over 80% of NoV infections worldwide. NoVs are highly infectious, very stable, and remarkably transmissible.[4, 5] Besides being a leading cause of epidemic acute gastroenteritis and an important cause of hospital acquired and sporadic infection, NoVs have also been described as a common etiology of travelers' diarrhea (TD).[6, 7] Interestingly, TD studies over time and in different locations have reported a marked diversity of GI- and GII-NoVs among cases.[6, 8-10] However, an almost equivalent number of NoV-associated travelers' diarrhea (NoV-TD) cases are associated with strains from both genogroups, which contrasts with the predominance of GII.4 viruses being associated with outbreak settings and sporadic cases worldwide.[11, 12]
The role of the antibodies elicited after a NoV infection, particularly those of specific antibody classes, has been difficult to discern as in some studies individuals with high prechallenge enzyme-linked immunosorbent assay (ELISA) titers were more likely to become infected than individuals with low preexisting titers.[13, 14] More recently, the presence of antibodies that block the binding of NoV virus-like particles (VLPs) to histo-blood group antigens (HBGAs) prior to challenge or inhibit VLP binding to red blood cells have correlated with protection against clinical NoV gastroenteritis.[15-17]
The aim of this study was to investigate the prevalence of NoV genogroup-specific antibodies in a group of US travelers to Mexico and the occurrence of natural NoV infection during travel. Recombinant GI [Norwalk virus (NV)] and GII [Houston virus (HOV)] VLPs were used to determine the role of preexisting serum antibodies [immunoglobulin A (IgA), IgG, IgM and HBGA-blocking antibodies] in protection against NoV-TD.
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The prevalence of NoV-associated TD found in this study was 16%, similar to what has been reported previously for comparable populations traveling to Mexico (circa 15%).[6, 9, 10] However, this result is an underestimate of the prevalence of NoV-TD in international travelers as we only studied diarrhea and not gastroenteritis with vomiting, a much more common presentation of illness caused by this group of viruses, and only half of the stool samples collected were available for testing. Similar to previous reports, GI-associated NoV-TD cases were more prevalent (75%) than GII-NoVs.[8, 9] This result supports previous studies confirming that NoVs are a commonly identified pathogen of TD in US travelers to Mexico.
To detect baseline levels of antibodies against NoVs among travelers, we chose to use VLPs from two different strains of NoV-VLPs (GI.1-NV and GII.4-HOV) that represent the predominant genotype within genogroups I and II, respectively. In this context, a significantly greater number of travelers had preexisting IgA antibodies to HOV-VLPs than to NV-VLPs (p = 0.025) and in higher titers (p = 0.0037). The higher seroprevalence rates for GII.4 may be due to the predominance of these strains worldwide. Although a confirmed history of previous NoV infection was not available for any of the study participants, serum samples from all study participants had IgG levels against NV- and HOV-VLPs above the assay cut-off, suggesting at least one previous exposure to the virus. The presence of preexisting antibodies against NoVs was not associated with protection specifically from GI or GII NoVs; however, the small number of test samples and the lack of testing of travelers who did not experience TD, limit this conclusion. Nonetheless, previous studies have demonstrated that a prior NoV infection does not confer long-term homologous protection against subsequent infections and disease.[13, 14]
Previous results from our laboratory and others suggest that antibodies capable of inhibiting the binding of VLPs to HBGAs or red blood cells have correlated with protection against disease.[15-17, 23] The frequency and the level of blocking antibodies found among travelers against HOV-VLP were higher than that against NV-VLP (p = <0.0001), in agreement with the serum IgA and IgG results. Of the 12 travelers infected with NoVs, 9 were considered seronegative for blocking antibodies (BT50 < 25) and 3 others had titers below the suggested threshold for protection against infection and viral illness (BT50 < 200). Future comprehensive studies are needed to confirm cross-reactivity of protection conferred by blocking antibodies.
Only 3 of 12 infected individuals had a fourfold or more rise in antibody titers following infection (data not shown). However, this difference was not attributed to the length of time between samples for both groups as individuals who showed a fourfold or more rise in antibody titers following infection had an average of 33 days between sample collection, similar to that of 29 days for individuals who did not have a significant sero-response. Nevertheless, these results could underestimate the antibody response in this population as the serological assays were carried out with VLPs representative of GI and GII viruses that may not have been homologous to the virus detected in stool, thus decreasing the sensitivity of the assay. For future studies, homologous VLPs for determining sero-responses to NoV infection should be used because of the high capsid sequence diversity observed in this group of viruses. Nonetheless, the DELFIA assay used in this study has previously been shown to be highly specific when compared with antigen detection in stool. The small quantity of stool samples was a limiting factor in carrying out genotyping assays in this study.
A high level of coinfections was observed among travelers with NoV-TD. This observation is in line with our previous studies in travelers where rates of coinfections are found to be over 60%. TD is characterized as multifactorial disease caused by a wide range of enteric pathogens including bacteria, viruses, and parasites. Furthermore, the ratio of pathogen coinfection in ill travelers is significant among the total number of cases. Other pathogens in addition to viruses must be sought for when defining the etiology of TD. However, it should be noted that the presence of copathogens did not appear to interfere with the stimulation of the immune response to NoVs as the travelers who showed a fourfold or more rise in antibody had multiple enteric pathogens detected in their stools.
In conclusion, we provide more evidence that NoVs are associated with the development of TD among US travelers to Mexico. Moreover, not only fewer travelers have antibodies against a GI-VLP compared with a GII-VLP, but also the amount of specific antibodies detected was lower for a GI-VLP than a GII-VLP. These results could partially explain the continuous presence of GI-NoV infections in travelers to these locations.[8, 9] Altogether, international travelers to developing regions of the world represent a unique and traceable population with relatively low prevalence of blocking antibodies and high rates of NoV endemicity, making them valuable for evaluation of NoV vaccines in the pipeline for development.